Human Cytomegalovirus (HCMV) is a herpesvirus that infects the majority of the population worldwide. HCMV establishes a life-long latent infection following a typically asymptomatic primary infection. Reactivation of HCMV from latency is a significant cause of morbidity and mortality in leukemia and lymphoma patients following bone marrow transplantation. The long-term goal of this research program is to identify the molecular mechanisms controlling HCMV latency and reactivation in bone marrow cells. To this end, an in vitro system using primary human hematopoietic cells has been developed that recapitulates HCMV latency. In this proposal, Aim 1 will identify viral cis- and trans-acting genetic determinants that control latency by creating recombinant viruses that lack candidate latency genes or sequences. These viruses will be analyzed for a defect in establishing latency or reactivating from latency in primary human hematopoietic cells using the in vitro model system. Candidate genetic determinants include regions of the genome that are unique to clinical strains of HCMV that can establish a latent infection. Further, distinct patterns of viral genes expressed in primitive hematopoietic cells that support an infection in vitro with the hallmarks of latency are candidate latency genes. In Aim 2, the cellular reservoir for HCMV latency in vivo will be identified by analyzing purified hematopoietic subpopulations from HCMV-seropositive individuals for the presence of HCMV genomes. Aim 3 will determine the ability of human hematopoietic progenitor cells infected in vitro to disseminate latently infected cells in immuncompromised mice following xenotransplantation. Human cells reconstituting the mouse blood system will be analyzed for the presence of HCMV genomes. These studies will elucidate the path by which HCMV genomes are disseminated into the circulating blood and begin to define key mechanisms governing HCMV latency in human hematopoietic cells, thereby identifying targets for treatment or prevention of HCMV disease in bone marrow transplant recipients.